Process for preparing (-)-(18-crown-6)-2,3,11,12-tetracarboxylic acid, and (-)-chiral stationary phases for resolution of racemic compounds using the same

ABSTRACT

The present invention relates to a process for preparing (−)-(18-crown-6)-2,3,11,12-tetracarboxylic acid and its use for (−)-chiral stationary phases for resolution of racemic compounds. More particularly, the present invention relates to the process for preparing (−)-(18-crown-6)-2,3,11,12-tetracarboxylic acid expressed by formula (1) and the use thereof as a stationary phases for resolution of racemic compounds, wherein the use of them provides excellent separation of a desired chiral compound from racemic mixture in employing capillary electrophoresis (CE) or liquid chromatography to elute the desired one first by controlling a flowing order of enantioners, thus allowing to be consistently separated in an economical due to much less requirement of eluent, quantitative and high purity manner.

This Application is a 317 of PCT/KR01/01926 filed Nov. 12, 2001.

FIELD OF THE INVENTION

The present invention relates to a process for preparing(−)-(18-crown-6)-2,3,11,12-tetracarboxylic acid and its use for(−)-chiral stationary phases for resolution of racemic compounds. Moreparticularly, the present invention relates to the process for preparing(−)-(18-crown-6)-2,3,11,12-tetracarboxylic acid expressed by thefollowing formula (1) and the use thereof as a stationary phases forresolution of racemic compounds, wherein the use of this stationaryphase provides excellent separation of a desired chiral compound fromracemic mixture in employing capillary electrophoresis (CE) or liquidchromatography (LC) to elute the desired one first by controlling aflowing order of enantiomers, thus allowing to be consistently separatedin an economical due to much less requirement of eluent, quantitativeand high purity manner,

wherein the compound of formula (1) is an enantiomer which has not beenreported for its preparation processes and uses.

On the other hand, (+)-isomer,(+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid, of the compound offormula (1) has been synthesized by Hiroyuki Nishi et al. and used forseparation of enantiomers from racemic mixtures (Journal ofChromatography A, 757, 1997, 225-235). Hiroyuki Nishi et al. have usedsaid enantiomer as a stationary phase of capillary electrophoresis orliquid chromatography to separate out a desired chiral compound fromracemic mixture which was unresolvable or difficult to resolvepreviously. This is particularly useful for the separation of aminocompounds.

Further, Yoshio Machida et al. have developed (+)-chiral stationaryphase for liquid chromatography by immobilizing(+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid on the surface of silicagel to separate enantiomers which was unresolvable or difficult toresolve previously (Journal of Chromatography A, 805, 1998, 85-92).

Recently, Myung Ho Hyun et al. have developed a chiral stationary phasefor liquid chromatography prepared by a different method immobilizing(+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid to silica gel to employin resolving various racemic mixtures (Korea Patent No. 262872; Journalof Chromatography A, 822, 1998, 155-161; Journal of Chromatography A,837, 1999, 75-82; Bull. Korean Chem. Soc. 1998, Vol. 19, No. 8,819-821).

In addition, it has been reported for resolution of racemic mixtureshaving amino acid or amine functional groups using(+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid in Journal ofChromatography A, 680, (1994) 253-261; Journal of Chromatography A, 685,(1994) 321-329; Anal. Chem. 1996, 68, 2361-2365; Journal ofChromatography A, 810 (1998) 33-41; Journal of Chromatography A, 666(1994) 367-373; Electrophoresis 1994, 15, 828-834; Journal ofChromatography A, 709 (1995) 81-88; Chromatographia Vol. 49, No. 11/12,(1999) 621-627; Chromatographia Vol. 33, No. 1/2, (1992) 32-36; Anal.Chem. 1992, 64, 2815-2820; Journal of Chromatography A, 716 (1995)371-379; Journal of Chromatography A, 715 (1995) 143-149; Journal ofChromatography A, 757 (1997) 328-332; Electrophoresis 1999, 20,2650-2655; Electrophoresis 1999, 20, 2605-2613.

Particular examples of amino compounds or drugs which exist in chiralmixtures include tyrosine, phenylglycilne, 2-amino-1-phenylethanol,normetanephrine, norephedrine, alanine-beta-naphthylamide, quinolonederivatives and the like. These optically pure chiral compound separatedfrom the racemic mixture have been possible for quantitative analysis.

However, in these days, technologies such as simulated moving bed (SMB)technology for efficient separation of optically pure compounds directlyfrom racemic mixtures in high yield and large scales are highlyincreased not only for quantitative analysis but also for thedevelopment the pharmaceutical and fine chemical industries (Chemical &Engineering New, 2000, Jun. 19). Development of novel chiral stationaryphases for production chiral drugs becomes significant.

In the process to obtain each enatiomer from racemic mixture in highyield and purity, first fractions are usually pure chiral compound butlater fractions are incomplete separation of the components. Thus, itmay result poor separation and low purity and yield for especially laterflowing chiral compound. Particularly, in the process to determineaccurate purity of chiral compound via quantitative analysis, if a majorchiral compound elutes first and a minor chiral one does later, itbecomes difficult to determine an accurate purity of later flowingcompound due to tailing effect of the first fractions. It is generalthat flowing concentration per unit time is lower and flowing time takeslonger for the later flowing compound than the first flowing one.

However, certain pharmaceutical compounds, known to provide effectivetreatment against disease states or to ameliorate medical conditions,often occur as a chiral mixture where one enantiomeric form has thedesired therapeutic activity whereas the other enantiomeric form of thesame compound causes undesirable side-effects and may limit drugefficacy. Therefore, it is highly beneficial to be able to separate outand collect the most effective forms of enantiomeric compounds.

Since FDA's (Food and Drug Administration's) Policy Statement revised in1992 for Development of New Drugs reported that each isomer of the samecompound having the same structure is regarded as a different compoundand side effects associated with undesired isomer in the human body haveoften reported, chiral separation with high yield and purity has becomemore and more important in pharmaceutical and fine chemical fields.

As a result, the demand in the development of capillary electrophoresis,LC chiral column, and simulated moving bed technologies to increaseseparation efficiency has been rapidly increased in recent years. Aseparation efficiency may be increased by controlling a flowing order ofchiral compounds to be separated. This flowing order in the SMBtechnology has a significant influence on relative difficulty of theprocess, purity of the separated compound, cost and the like.

Inventors have applied ASTEC's Cyclobond™, Chirobiotic™ V, T, and Rcolumns which is based on bonding α-, β- or γ-cyclodextrin, orvancomycin to silica gel to separate various chiral compounds. However,the process is inefficient because it requires too complicate processand much efforts, even though it provides well separation of eachenantiomer.

In the process to separate (R) and (S)-isomer and determine accuratepurity thereof by employing capillary electrophoresis or LC chiralstationary phase column, if the first flowing fractions are major andthe later ones are minor, the first flowing ones can be eluted with someof the later flowing ones due to tailing effect of the first flowingones, thus it becomes difficult to obtain accurate optical purity. Inthis case, if the minor compound can be eluted first and major onelater, it can avoid tailing effect of the major compound, thus allowingto determine an accurate optical purity of the minor compound. Eventhough there is tailing effect of the minor compound, the tailing effectassociated with the minor compound must be much smaller than that withthe major one or can be ignored, thus it does not affect to determineaccurate optical purities. In case that the large scale chiralseparation is performed by using SMB column or LC chiral stationaryphase column, it will be preferable to elute a desired chiral compoundfirst to avoid tailing effect of the later flowing compound, furtherprovides several advantages in reducing amount of eluent, performingprocess and the processing time. Further, the control of the flowingorder allows more accurate optical purity.

SUMMARY OF THE INVENTION

The inventors have completed the present invention by providing aprocess for preparing (−)-(18-crown-6)-2,3,11,12-tetracarboxylic acidand its use for (−)-chiral stationary phases for resolution of racemiccompounds to control flowing order in the chiral separation usingcapillary electrophoresis or LC chiral column and producing theoptically pure compound effectively.

Accordingly, an object of the present invention is to provide a processfor preparing (−)-(18-crown-6)-2,3,11,12-tetracarboxylic acid. Anotherobject is to provide its use for (−)-chiral stationary phases forresolution of racemic compounds and preparation thereof. Further objectis to provide its use in capillary electrophoresis.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a process for preparing(−)-(18-crown-6)-2,3,11,12-tetracarboxylic acid of formula (1) to beused for resolution of racemic compounds.

The present invention also provides (−)-chiral stationary phases offormula (2) for LC chiral column and manufacturing method thereof,

wherein R represents a hydrogen atom or C₁-C₄ low alkyl group; each ofZ₁, Z₂, and Z₃ represents CO₂H or a complex of formula (3) bonded withsilica gel,

wherein R is same as defined previously.

The present invention will be discussed in more detail hereunder.

(+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid has been utilized as acapillary electrophoresis or chiral stationary phase by liquidchromatography (HPLC) as described above in the prior art. On the otherhand, (−)-(18-crown-6)-2,3,11,12-tetracarboxylic acid of formula (1) ofthe present invention is a different compound from the known isomer,(+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid, and has differentutilities. For example, in order to obtain (R)-isomers of amino acids,which do not naturally exist, by chiral separation SMB method or(−)-chiral stationary phase column unlike(+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid is used to elute(R)-isomer first, thus providing several advantages in avoiding toreduce optical purity of (R)-isomer, shortening the chiral separationprocess with saving time, and reduction in cost.

As shown in Scheme 1, (−)-(18-crown-6)-2,3,11,12-tetracarboxylic acid offormula (1) is prepared by hydrolysis of octaethyl(−)-(18-crown-6)-2,3,11,12-tetracarboxamide of formula (6) obtained bycondensation of N,N,N′,N′-tetraalkyl-D-tartaramide of formula (4) withthe compound of formula (5),

wherein R′ represents C₁-C₄ low alkyl; X represents Cl, Br, I,p-toluenesulfoxide(TsO) or methanesulfoxide(MsO).

N,N,N′,N′-Tetraalkyl-D-tartaramide of formula (4) dissolved in anorganic solvent chosen from dimethylformamide, dimethylacetamide, andtetrahydrofuran is reacted with a base chosen from sodium hydride,thallium ethoxide, and potassium t-butoxide. 1-20 Equivalents of thecompound of formula (5) is added and condensed at 50-100° C. to produceoctaethyl (−)-(18-crown-6)-2,3,11,12-tetracarboxamide of formula (6).The obtained octaethyl (−)-(18-crown-6)-2,3,11,12-tetracarboxamide offormula (6) is hydrolyzed in acidic solution at 50-100° C. to produce(−)-(18-crown-6)-2,3,11,12-tetracarboxylic acid of formula (1).

The present invention further provides a (−)-chiral stationary phase forliquid chromatography using (−)-(18-crown-6)-23,11,12-tetracarboxylicacid of formula (1) and a process for preparing the same. (−)-Chiralstationary phase is prepare by converting(−)-(18-crown-6)-2,3,11,12-tefracarboxylic acid of formula (1) to thecorresponding anhydride of formula (7) and then condensing the resultwith aminopropyl silica gel or monoalkylaminopropyl silica gel, or bycondensing the result directly with aminopropyl silica gel ormonoalkylaminopropyl silica gel using a binding agent,

wherein each of R, Z₁, Z₂ and Z₃ are same as defined previously.

In Scheme 2, (−)-(18-crown-6)-2,3,11,12-tetracarboxylic acid of formula(1) is treated with acetyl chloride or acetic anhydride, thionylchloride, phosphorus oxychloride without using any organic solvent; with2,2′-dipyridyl disulfide or 4,4′-dipyridyl disulfide in the presence oftrialkylphosphine or triphenylphosphine in an organic solvent chosenfrom dichloromethane, dichloroethane, acetone, toluene, benzene, etherand ethyl acetate; or with phosphorus pentachloride in the same solventsystem to produce the corresponding anhydride of formula (7). Theobtained anhydride of formula (7) is condensed with aminopropyl silicagel or monoalkylaminopropyl silica gel in the presence of triethylamineor pyridine in an organic solvent chosen from dichloromethane,dichloroethane, toluene, and benzene to produce (−)-chiral stationaryphase of formula (2).

And also (−)-chiral stationary phase of formula (2) may be prepared bycondensing (−)-(18-crown-6)-2,3,11,12-tetracarboxylic acid of formula(1) with aminopropyl silica gel or monoalkylaminopropyl silica gel inthe presence of 2 equivalents of 1,3-dicyclohexylcarbodiimide orN-ethoxycarbonyl-2-ethoxy-1,2-dihydroquiloline as a binding agent in anorganic solvent chosen from dichloromethane, dichloroethane, acetone,toluene, benzene, ether and ethyl acetate.

The (−)-chiral stationary phase of formula (2) is suspended in methanoland the slurry is charged in HPLC column using slurry charger to produce(−)-chiral column.

The following examples and experimental examples are intended to furtherillustrate the present invention without limiting its scope.

EXAMPLE 1

Preparation of Octamethyl (−)-(18-crown-6)-2,3,11,12-tetracarboxamide

N,N,N′,N′,-tetramethyl-D-tartaramide (35 g) was added regularly insodium hydride (8.02 g, 95%) in dimethylformamide under N₂, whilestirring at 0° C. After the reaction mixture was further stirred at roomtemperature for 1 hour, di(ethylene glycol)di-p-tosylate (71.05 g)dissolved in dimethylformamide was added to it. After performing thereaction at 80° C. for 8 hours, dimethylformamide was removed under thepressure. The residue was triturated with chloroform and the precipitatewas filtered out. The filtrate was evaporated under the pressure and theresidue was purified by column chromatography on alumina by eluting withdichloromethane to yield the desired product (6.5 g).

¹H NMR(CDCl₃) δ(ppm): 2.92(s, 12H), 3.16(s, 12H), 3.65-3.96(m, 16H),4.80(s, 4H); [a]_(D)=−110°(c=1.5, CHCl₃)

EXAMPLE 2

Preparation of Octamethyl (−)-(18-crown-6)-2,3,11,12-tetracarboxamide

Thallium ethoxide (42.5 g) was added regularly intoN,N,N′,N′,-tetramethyl-D-tartaramide (17.5 g) in dimethylformamide underN₂, while stirring at 0° C. After the reaction mixture was furtherstirred at room temperature for 1 hour, bromoethyl ether (200 g) wasadded to it. After performing the reaction at 80° C. for 8 hours,dimethylformamide and excess bromoethyl ether were removed under thepressure. The residue was dissolved in dichloromethane, washed withwater, and evaporated under the pressure. The residue was purified bycolumn chromatography on alumina by eluting with dichloromethane. Theobtained fractions were evaporated and the result was dissolved indimethylformamide. In another flask, thallium ethoxide (21.2 g) wasadded regularly into N,N,N′,N′,-tetramethyl-D-tartaramide (8.8 g) indimethylformamide under N₂, while stirring at 0° C. After the reactionmixture was further stirred at room temperature for 1 hour, the obtainedcompound previously dissolved in small amount of dimethylformamide wasadded to the reaction mixture. After performing the reaction at 80° C.for 8 hours, dimethylformamide was removed under the pressure. Theresidue was triturated with chloroform and the precipitate was filteredout. The filtrate was evaporated under the pressure and the residue waspurified by column chromatography on alumina by eluting withdichloromethane to yield the desired product (6.0 g).

¹H NMR(CDCl₃) δ(ppm): 2.92(s, 12H), 3.16(s, 12H), 3.65-3.96(m, 16H),4.80(s,4); [a]_(D)=−108°(c=1.5, CHCl₃)

EXAMPLE 3

Preparation of (−)-(18-crown-6)-2,3,11,12-tetracarboxylic Acid

2.5N of hydrochloride solution (60 mL) was added to octamethyl(−)-(18-crown-6)-2,3,11,12-tetracarboxamide (6 g) and the mixture wasstirred at 80° C. for 24 hours. The reaction mixture was evaporatedunder the pressure and the residue was past through an ion exchangeresin by using water. Water was evaporated and the residue wascrystallized using minimum amount of water to yield the desired product(3.9 g).

¹H NMR(CD₃OD) δ(ppm): 3.63-3.91(m, 16H), 4.65(s, 4H); [α]_(D)=−63°(c=1,MeOH)

EXAMPLE 4

Preparation of (−)-(18-crown-6)-2,3,11,12-tetracarboxylic anhydride

Acetyl chloride (24 mL) was added to(−)-(18-crown-6)-2,3,11,12-tetracarboxylic acid (969 mg) under N₂ andthe reaction mixture was heated at reflux for 18 hours. Excess acetylchloride was evaporated under the pressure to yield the desired product(890 mg).

¹H NMR(CDCl₃) δ(ppm): 3.62-4.17(m, 16H), 4.83(s, 4H)

EXAMPLE 5

Preparation of (−)-chiral Stationary Phase (Compound 2; R=H)

Dichloromethane (33 mL) and triethyl amine (0.77 mL) were added toaminopropyl silica gel (8.08 g) under N₂.(−)-(18-Crown-6)-2,3,11,12-tetracarboxylic anhydride, obtained inExample 4, dissolved in dichloromethane (17 mL) was added to thereaction mixture at 0° C. The reaction mixture was stirred at roomtemperature for 24 hours, filtered, washed with methanol, water,methanol, dichloromethane, and hexane in series and dried to yield thedesired product (8.8 g).

EXAMPLE 6

Preparation of (−)-chiral Stationary Phase (Compound 2; R=Me)

(−)-Chiral stationary phase was prepared by the same procedure as thatof Example 5, excepting using monomethylaminopropyl silica gel insteadof aminopropyl silica gel to yield the desired product (8.8 g).

EXAMPLE 7

Preparation of (−)-chiral Stationary Phase (Compound 2; R=H)

To a mixture of (−)-(18-crown-6)-2,3,11,12-tetracarboxylic anhydride(969 mg), 1,3-dicyclohexylcarbodiamide (908 mg) and aminopropyl silicagel (8.08 g) was added benzene (33 mL). The reaction mixture was heatedat reflux for 4 hours, filtered, washed with methanol, water, methanol,dichloromethane, and hexane in series and dried to yield the desiredproduct (8.8 g).

EXAMPLE 8

Preparation of (−)-chiral Stationary Phase (Compound 2; R=Me)

(−)-Chiral stationary phase was prepared by the same procedure as thatof Example 7, excepting using monomethylaminopropyl silica gel insteadof aminopropyl silica gel to yield the desired product (8.8 g).

Experimental Example 1

Chiral separation by (-)-chiral column IC charged with (−)-chiralstationary phase (compound 2; R =H)

The (−)-chiral stationary phase (2.5 g) prepared in Example 5 wassuspended in methanol (20 mL) and charged into HPLC column (150 mm ×4.6mm I.D.) by using slurry charger to produce (−)-chiral column.Separation of the following pounds in Table 1 was performed by using theprepared (−)-chiral column with eluent of methanol/water =80/20 andsulfuric acid (10 mM) under the condition of low rate of 0.5 mL/min (1.2mL/min for quinolone A and B), detector of 210 nm UV (294 m forquinolone A and B), and a temperature of 20° C. The result was comparedwith that performed the (+)-chiral stationary phase liquidchromatography prepared by using(+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid and summarized in Table1.

TABLE 1 (−)-chiral column (+)-chiral column First fraction Secondfraction First fraction Second fraction Racemic Capacity AbsoluteCapacity Absolute Capacity Absolute Capacity Absolute compound factor k1configuration factor k2 configuration factor k1 configuration factor k2configuration Alanine 1.36 R 1.76 S 1.37 S 1.76 R Tyrosine 0.85 R 1.21 S0.84 S 1.21 R Threonine 0.24 2S, 3R 0.35 2R, 3S 0.24 2R, 3S 0.34 2S, 3RTocainide 1.91 R 2.21 S 1.90 S 2.22 R 1-Aminoindane 1.17 S 1.80 R 1.16 R1.79 S 2-Phenylglycinol 1.43 R 1.95 S 1.44 S 1.95 R Qunolone A 9.50 R10.95 S 9.50 S 10.94 R Qunolone B 14.02 R 14.74 S 14.01 S 14.76 R

Experimental Example 2

Chiral Separation by (−)-chiral Column LC Charged with (−)-chiralStationary Phase (Compound 2; R=Me)

The (−)-chiral stationary phase (2.5 g) prepared in Example 6 wassuspended in methanol (20 mL) and the slurry was charged into HPLCcolumn (150 mm×4.6 mm I. D.) by using slurry charger to produce(−)-chiral column. Separation of the following compounds in Table 2 wasperformed by using the prepared (−)-chiral column with eluent ofmethanol/water=80/20 and sulfuric acid (10 mM) under the condition offlow rate of 1.2 mL/min, detector of 210 nm UV (294 nm UV for quinoloneB), and a temperature of 20° C. The result was compared with thatperformed with (+)-chiral stationary phase liquid chromatographyprepared by using (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid andsummarized in Table 2.

TABLE 2 (−)-chiral column (+)-chiral column First fraction Secondfraction First fraction Second fraction Racemic Capacity AbsoluteCapacity Absolute Capacity Absolute Capacity Absolute compound factor k1configuration factor k2 configuration factor k1 configuration factor k2configuration Leucine 5.98 R 7.42 S 5.99 S 7.43 R Phenylglycine 11.13 R19.61 S 11.14 S 19.61 R Tocainide 1.52 R 1.81 S 1.52 S 1.82 R1-Aminoindane 8.31 S 20.13 R 8.32 R 20.13 S 2-Phenylglycinol 14.71 R17.94 S 14.70 S 17.93 R Qunolone B 12.80 R 20.94 S 12.81 S 20.92 R

Experimental Example 3

Chiral Separation by (−)-chiral Column LC Charged with (−)-chiralStationary Phase (Compound 2; R=H)

Separation of the following compounds in Table 3 was performed by usingthe prepared (−)-chiral column charged with (−)-chiral stationary phase(compound 2; R=H) with eluent of methanol/water=80/20 and sulfuric acid(10 mM) under the condition of flow rate of 1 mL/min (2 mL/min forquinolone 1 and 2), detector of 210 nm UV (294 nm for quinolone 2), anda temperature of 20° C. The result was compared with that performed with(+)-chiral stationary phase liquid chromatography prepared by using(+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid and summarized in Table3.

TABLE 3 (−)-chiral column (+)-chiral column Racemic Optical purityAbsolute Optical purity Absolute compound Yield (mg) (% ee)configuration Yield (mg) (% ee) configuration Tyrosine 9.7 >99.9 R 7.399.7 R 1-Aminoindane 9.7 >99.9 S 7.4 99.7 S 2-Phenylglycinol 9.7 >99.9 R7.1 98.8 R Qunoline A 9.3 99.8 R 5.5 98.0 R

Experimental Example 4

Chiral Separation by (−)-chiral Column LC Charged with (−)-chiralStationary Phase (Compound 2; R=Me)

Separation of the following compounds in Table 4 was performed by usingthe prepared (−)-chiral column charged with (−)-chiral stationary phase(compound 2; R=Me) with eluent of methanol/water=80/20 and sulfuric acid(10 mM) under the condition of flow rate of 2.5 mL/min, detector of 210nm UV (294 nm UV for quinolone 1), temperature of 20° C. The result wascompared with that performed with (+)-chiral stationary phase liquidchromatography prepared by using(+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid and summarized in Table4.

TABLE 4 (−)-chiral column (+)-chiral column Racemic Yield Optical purityAbsolute Yield Optical purity Absolute compound (mg) Yield (%) (% ee)configuration (mg) Yield (%) (% ee) configuration Tyrosine 9.7 97 >99.9R 8.2 82 99.0 R 1-Aminoindane 9.9 99 >99.9 S 9.7 97 99.8 S2-Phenylglycinol 9.7 97 >99.9 R 8.5 85 98.8 R Qunolone B 9.9 99 99.8 R9.7 97 99.5 R

As shown the above Examples and Experimental Examples, it is noted thatthe present invention provides uses of(−)-(18-crown-6)-2,3,11,12-tetracarboxylic acid of formula (1) in chiralseparation by capillary electrophoresis and (−)-chiral stationary phasefor liquid chromatography. The present invention further providesexcellent separation efficiency in high yield and high purity by elutingthe desired chiral compound first, and allows determining accurateoptical purity thereof.

What is claimed is:
 1. A process for preparing(−)-(18-crown-6)-2,3,11,12-tetracarboxylic acid of formula (1) byhydrolysis of octaethyl (−)-(18-crown-6)-2,3,11,12-tetracarboxamide offormula (6) obtained by condensation ofN,N,N′,N′-tetraalkyl-D-tartaramide of formula (4) with the compound offormula (5),

wherein R′ represents C₁-C₄ low alkyl; X represents Cl, Br, I,p-toluenesulfoxide(TsO) or methanesulfoxide(MsO).
 2. A (−)-chiralstationary phase of formula (2) for liquid chromatography for resolutionof chiral compound from racemic mixtures,

wherein R represents a hydrogen atom or C₁-C₄ low alkyl group; each ofZ₁, Z₂, and Z₃ represents CO₂H or a complex of formula (3) bonded withsilica gel,

wherein R is same as defined previously.
 3. A process for preparing(−)-chiral stationary phase of formula (2) by converting(−)-(18-crown-6)-2,3,11,12-tetracarboxylic acid of formula (1) to thecorresponding anhydride of formula (7) and condensing the result withaminopropyl silica gel or monoalkylaminopropyl silica gel,

wherein R represents a hydrogen atom or C₁-C₄ low alkyl group; each ofZ₁, Z₂ and Z₃ represents CO₂H or a complex of formula (3) bonded withsilica gel.

wherein R is same as defined previously.
 4. A process for preparing(−)-chiral stationary phase of formula (2) by condensing(−)-(18-crown-6)-2,3,11,12-tetracarboxylic acid of formula (1) withaminopropyl silica gel or monoalkylaminopropyl silica gel using abinding agent,

wherein R represents a hydrogen atom or C₁-C₄ low alkyl group; each ofZ₁, Z₂, and Z₃ represents CO₂H or a complex of formula (3) bonded withsilica gel,

wherein R is same as defined previously.
 5. A (−)-chiral column forliquid chromatography prepared by using (−)-chiral stationary phase offormula (2),

wherein R represents a hydrogen atom or C₁-C₄ low alkyl group; each ofZ₁, Z₂, and Z₃ represents CO₂H or a complex of formula (3) bonded withsilica gel,

wherein R is same as defined previously.